Protein crystallization has three major applications: (1) structural biology and drug design, (2) bioseparations, and (3) purification. Crystal formation is a slow, and tedious process typically based on preparation of a saturated protein solution following nucleation, at optimal conditions. Optimal conditions for protein crystallization include optimal protein concentration in the saturated solution, pH and temperature among others whereas these conditions are determined by extensive trial and error experiments.
Conventional protein crystallization methods and methods for determining the conditions that facilitate protein crystallization are disclosed in U.S. Pat. Nos. 6,596,077; 6,593,118; 6,579,358; 6,500,933; 6,409,832; 6,268,158; 5,976,325; 5,728,559; 5,271,795; 5,104,478; 4,737,232 and 4,330,363 among others.
Crystallization of bio-molecules, specifically proteins, in gels such as Silica hydrogel, agarose and polyacrylamide, is known in the art (e.g. CrystalEx™, Hampton Research Corp.). This method is considered advantageous since it is devoid of considerations of buoyancy, convection and sedimentation as compared with growth from liquid solutions. This method further regulates molecular diffusion, by the viscosity of the gel, and hence mimics in many respects the beneficial properties of crystal growth in microgravity environment like space. Another advantage of this method is that the crystals are maintained encapsulated in the gel at ambient temperature and in that configuration can even be directly subjected to the X-ray diffraction. This method basically involves adding a protein solution at concentrations of 10-20 mg/ml to a gel either prior to polymerization or following polymerization following storage within the gel at a controlled temperature for days to weeks, until protein crystallization takes place. It was shown that using this method, appropriate crystals were obtained (Sauter et al., Proteins: Structure, Function and Genetics 48:146-150, 2002), however, this method is rarely employed for protein crystal growth and is as slow as the other methods known in the art.
Isoelectric focusing (IEF) technique is widely used for protein separation and purification on the basis of their characteristic net electrical charge that varies with pH. Proteins are subjected to an electric field in a pH gradient wherein each protein migrates to a point within the gradient at which its net charge is zero, this point is called “the isoelectric point” or “pI”.
IEF techniques for protein separation and purification are described in WO03/008977, by the inventor of the present invention, U.S. Pat. Nos. 6,537,432; 5,480,562; 5,464,517; 5,451,662; 5,082,548; 4,971,670; 4,495,279; and 4,243,507 among others.
There is an unmet need to overcome the obstacles encountered with crystal growth and to provide more rapid and less exhaustive means for enabling crystallization, particularly, protein crystallization.